Method for transdifferentiation of body tissues

ABSTRACT

The invention relates to methods for transdifferentiation of body tissues which can be used to generate specific cell types needed for regenerating organs or body parts, following cellular degeneration, injury or amputation. The present invention also describes the use of tissue transdifferentiation for treating cancer and autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending Ser. No. 10/600,745which is a divisional of Ser. No. 09/856,881, now U.S. Pat. No.6,671,397 issued Dec. 30, 2003. All of said applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Multiple invertebrates and several vertebrate species are known topossess the ability to regenerate lost body parts (Goss, Clin. Orthop.,1980, 151:270-282; Kawamura and Fujiwara, Sem. Cell Biol., 1995,6:117-126; Tsonis, Devel. Biol., 2000, 221:273-284). Thus, invertebratescan reconstruct the whole body from small pieces (Kawamura and Fujiwara,supra). Examples of regeneration in vertebrates include (i) rabbits andbats which can fill in holes punched through their ears; (ii) adultsalamanders which can regenerate a complete limb after amputation; and(iii) mice which can replace the tip of a foretoe when it is amputateddistal to the last joint (Goss and Grimes, Am. Zool., 1972, 12:151;Neufeld and Zhao, pp. 243-252, in: Limb Development and Regeneration,Fallon ed., John Wiley and Sons, 1993).

In humans, the fingertips of young children have also been shown toregrow after amputation distal to the last joint (Goss, supra;Illingworth, J. Ped. Surg., 1974, 9:853-858). Two factors have beenshown to be important for this regeneration: (1) the opened surface of afresh wound that can be covered by epidermal epithelium originating fromthe margins of the amputation site (Stocum, pp. 32-53, In: Regulation ofVertebrate Limb Regeneration, Sicard ed., Oxford Univ. Press, 1985), and(2) an adequate nerve supply at the wound surface (Singer et al. Anat.Embryol., 1987, 177:29-36).

The cellular mechanisms underlying regeneration have been studied for anumber of years, and there appear to be some conserved features betweenspecies. In vertebrates, there are two ways in which regenerationoccurs. In some tissues, multipotent quiescent stem cells becomeactivated by damage and proliferate to produce new cells of severaldifferent terminally differentiated phenotypes. Alternatively, there maybe a change in the phenotype of the functional, fully differentiatedcells, such that they lose many of their differentiated characteristics,and proliferate to form new fully differentiated cells of otherphenotype. This latter process has been termed Atransdifferentiation@(Okada, pp. 349-380, in: Current Topics in Developmental Biology,Denis-Donini et al. eds., Acad. Press, 1980; Okada,Trans-differentiation, Oxford Sci. Publ., 1991).

Retinal regeneration represents an example of the regenerative processthat can occur either through stem cells or via transdifferentiation;depending on the species. Thus, teleost fish contain a population ofretinal progenitor stem cells that can act as a source of new retinalneurons following damage (Hitchcock and Raymond, Trends Neurosci., 1992,15:103-108). In contrast, amphibians and embryonic chicks can regeneratetheir retina by a process that involves transdifferentiation of thecells in the pigment epithelium (RPE) to neural retinal progenitors (Rehand Pittack, Sem. Cell Biol., 1995, 6:137-142).

The existence of regeneration by transdifferentiation was questioned fora long time as it was not consistent with the classic view ofdifferentiation, according to which a once acquired cellular phenotypewas considered to be fixed due to irreversible changes in the geneexpression pattern. However, the development of in vitro cell culturesystems allowed the unequivocal experimental demonstration ofregeneration by transdifferentiation. Thus, it has been shown thatcultured fully differentiated pigmented epithelial cells of adult newtiris have the ability to dedifferentiate and proliferate to form a newtissue, lens (Eguchi et al., Proc. Natl. Acad. Sci. USA, 1974,70:5052-5056; Abe and Eguchi, Dev. Growth Diff., 1977, 19:309-317).

Both in vivo and in vitro studies have demonstrated that cytoplasmicsignals and changes in the gene expression (e.g., selective geneactivation and/or silencing) caused by interactions with growth factorsand components of the extracellular matrix are important in the controlof cellular transdifferentiation (Kodama and Eguchi. Sem. Cell Biol.,1995, 6:143-149; Rao and Reddy, ibid., 151-156). Thus, it has been shownthat copper deficiency in rats leads to loss of cell-cell interactions,altered microenvironment and global apoptosis of acinar cells in thepancreas which, in turn, causes oval and ductal pancreatic cells toundergo active proliferation resulting in their transdifferentiationinto liver hepatocytes (Rao and Reddy, supra). In another series ofexperiments conducted with the neural crest-derived pigmented skin cells(chromatophores) of the Axolotl (Ambystoma mexicanum), it has been shownthat the addition of guanosine can cause these cells totransdifferentiate from one pigmented cell type to another (Frost etal., Pigm. Cell Res., 1987, 1:37-43; Thibaudeau and Holder, Pigm. CellRes., 1998, 11:38-44).

It is believed that the replacement of complex appendages (i.e.,epimorphic regeneration) following amputation in lower vertebrates alsooccurs by transdifferentiation (Goss, supra). Thus, during epimorphicregeneration, epidermal wound healing is followed by the accumulation ofdedifferentiated blastemal cells beneath the wound epidermis. Theseblastemal cells are thought to originate by the dedifferentiation of themesenchymal and Schwann cells of the stump tissue (Brockes, Science,1984, 225:1280-1287) which then redifferentiate to reconstruct the limbtissue (Singer et al., Anat. Embyol., 1987, 177:29-36).

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods for inducing mammaliancells to transdifferentiate and to uses of such cells. Cells whichdisplay morphological and functional characteristics representative ofterminal differentiation are induced to change into other cell types.These cells may be derived from a plurality of organisms and from anybody tissue.

In one aspect, the present invention provides a method fortransdifferentiating mammalian cells comprising the steps of:

(a) contacting said cells with an effective amount for dedifferentiationof an agent which causes dedifferentiation of said cells, producingdedifferentiated cells;

(b) contacting said dedifferentiated cells of step (a) with an amounteffective for transdifferentiation of an agent which causestransdifferentiation of said dedifferentiated cells;

(c) contacting said cells from step (b) with an amount effective forstabilization of an agent which causes stabilization of cells producedin step (b); and

(d) recovering stabilized, transdifferentiated cells.

In another aspect, the present invention provides a method forregenerating or creating a developmental field in a remnant of astructure in a mammal, said structure having been partially destroyed,comprising the steps of:

(a) dedifferentiating said remnant of said structure;

(b) transdifferentiating said remnant of said structure of step (a); and

(c) stabilizing said remnant of said structure of step (b); therebycreating a developmental field in said remnant.

In yet another aspect, the present invention provides a method fortreating cancer in a mammal comprising contacting said cancer with anamount or an agent effective to cause transdifferentiation of saidcancer into benign cells.

In a further aspect, the present invention, the present inventionprovides a method for inhibiting the progression of an antibody-mediatedautoimmune disease in a patient comprising the steps of:

(a) obtaining cells from said patient of the type which are underautoimmune attack;

(b) contacting said cells with an amount of a transdifferentiation agenteffective to convert said cells to a normal phenotype;

(c) culturing said converted cells in vitro to amplify said cells;

(d) immobilizing said cells on a membrane which allows blood to enterbut retains the cell; and

(d) contacting said immobilized cells with said patients' blood, therebyremoving said antibodies from said patients' blood.

In a still further aspect, the present invention provides a method forregenerating a tissue or organ in the body of a mammal, wherein saidtissue or organ is damaged due to injury or is missing, comprising thesteps of:

(a) dedifferentiating the cells at the site of injury by administering adedifferentiating-effective amount of a dedifferentiating agent;

(b) transdifferentiating said dedifferentiated cells of step (a) bycontacting said cells with a transdifferentiating-effective amount of atransdifferentiation agent; and

(c) stabilizing the transdifferentiated cells of step (b) by contactingsaid cells with stabilization-effective amount of stabilizing agent.

In a still further aspect, the present invention provides a method forproducing stem cells comprising the steps of:

(a) obtaining melanocytes from a patient's skin cells;

(b) contacting said melanocytes with an agent which causestransdifferentiation; and

(c) recovering stem cells.

In a still further aspect, the present invention proves a method forproducing stem cells comprising the steps of:

(a) obtaining melanocytes from a patient's skin cells;

(b) contacting said melanocytes with an agent which causestransdifferentiation for a time and at a concentration effective toproduce stem cells; and

(c) recovering stem cells.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron micrograph taken from an Axolotl which was fed GMP(described in Example 5B). On the left is a process of a xanthophore. Onthe right is a process of a melanophore. pt=pterinosome; m=melanosome;p=premelanosome; * marks artifacts due to tears in the plastic. Themelanosomes are oval or round structures build on an internal lattice,and partially composed of numerous small round bodies as seen in themarked premelanosome. The pterinosomes appear as vacuoles with wispy orconcentric fibrillar material.

FIG. 2 is the same animal as in FIG. 1. Pterinosomes and melanosomes areseen in the same cell. Furthermore, a hybrid organelle is seen at thebottom of the picture. This cell is judged to be transdifferentiatingbased on the following criteria: (a) presence of numerous pterinosomesand melanosomes in the same cell, and (b) presence of some organelleswith characteristics suggestive of both melanosomes and pterinosomes.

FIG. 3 is a blow-up of the hybrid organelle seen at the bottom of FIG.2.

FIG. 4 is a Gomori trichrome stained section of an Axolotl which was fedGMP (described in Example 5B). E=epidermis; C=collagen band in dermisjust below basement membrane; M=melanocyte. The two arrows mark the cellwhich is transdifferentiating from a melanocyte into a xanthophore. Thesmall arrow points to melanosomes. The larger arrow points to apterinosome, which appears as a white vacuole.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents, and literature references cited hereinare hereby incorporated by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

Definitions

As used herein, the following terms are defined for purposes of thisinvention.

ATransdifferentiation@ refers to the capacity of differentiated cells ofone type to lose identifying characteristics and to change theirphenotype to that of other fully differentiated cells.

ACell destabilization@ or Adedifferentiation@ refers to the loss ofphenotypic characteristics of a differentiated cell by activating ordeactivating genes or metabolic pathways.

ACell stabilization@ refers to the maintenance of phenotypiccharacteristics of a differentiated cell by maintaining, activating ordeactivating genes or metabolic pathways.

AMorphogenetic field@ refers to a group of cells which have the capacityto give rise to a particular organ (e.g., pancreas, liver) or appendage(e.g., limb, tail) during embryogenesis or subsequent regeneration. Itcan be alternatively called a Adevelopmental field,@ or Aepimorphicfield,@ or Aprimary field@ (Hopper and Hart, Foundations of AnimalDevelopment, Oxford Univ. Press, 1980, p. 314).

AToxin@ refers to a substance which is poisonous to a cell. A toxin mayor may not be a protein. Non-limiting examples of toxins include: (a)heavy metals such as cadmium, copper and zinc; (b) strong acids or basessuch as hydrochloric acid (<pH 5) or sodium hydroxide (>pH 8); (c) ATPinhibitors, such as ATPase, and (d) poisons that destabilize membranes,such as detergents.

The Aneural crest@ refers to ectoderm-derived cells which duringdevelopment of the embryo are found interposed between the neural tubeand the ectoderm (LeDouarin, The Neural Crest, Cambridge Univ. Press,1982). Cells of the embryonic neural crest give rise to a wide range oftissues which include: (a) cells of the peripheral nervous system; (b)dermal bone; (c) cephalic connective tissue; (d) pigment cells; (e)calcitonin secreting cells; (f) meninges; (g) Schwann cells; (h)odontoblasts, and (i) adrenal medulla.

AChromatophores@ refers to specialized cells for animal coloration whichare usually of neural crest origin. Each of the chromatophores has apigment containing organelle derived from the endoplasmic reticulum.There are three classes of chromatophores that are defined based on thetype of pigment contained within these organelles: melanophores,xanthophores, and iridiophores (Ide, Curr. Topics Dev. Biol., 1986,20:79-87; Bagnara, The Neural Crest as a Source of Stem Cells, pp.57-87, In: Developmental and Evolutionary Aspects of the Neural Crest,Maderson ed., John Wiley and Sons, 1987).

AMelanophores@ are chromatophores which contain the pigment melanin. Themelanin is contained in organelles called melanosomes.

AXanthophores@ are yellow, orange or red colored chromatophores. Thepigments of xanthophores are a class of cyclic compounds calledpteridines. Pteridines, which are derived from guanosine triphosphate,are contained in an organelle called a pterinosome. Xanthophores alsosometimes contain an organelle called a carotenoid droplet.

Alridophores@ contain a pigment composed of crystalline deposits ofpurines. The crystalline deposits are contained in organelles calledreflecting platelets, so named because the organelles scatter andreflect light.

ARemnant@ is a portion of a structure which remains after damage byamputation, disease or other agents.

APartially destroyed@ refers to a structure (e.g. tissue organ orappendage, such as a limb) in which (a) a percentage of the structure=smass is removed, or b) the internal pattern and numbers of cellscomprising the structure are damaged or killed while some vestigal cellsand/or pattern remains.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

It is envisioned that the present invention will be used to producecells by transdifferentiation for the replacement of body tissues,organs, components or structures which are missing or damaged due totrauma, age, metabolic or toxic injury, disease, idiopathic loss, or anyother cause.

In another aspect, transdifferentiation is used to regenerate externalstructures such as fingers, toes or parts of such structures.

In still another aspect, transdifferentiation is used to treat cancer bymaking cancer cells adopt non-malignant phenotypes.

Finally, according to the present invention transdifferentiation is usedto provide autologous cells for removal of harmful auto-antibodies.

Without wishing to be bound by theory, it is believed that the methodsdetailed for (1) regeneration of body parts and (2) transdifferentiationof tissues/cells represent two complementary, intertwined, andindispensable aspects of a biological entity which is termed aAmorphogenetic field.@ The methods herein described cause reconstitutionand, in some cases, creation, of morphogenetic fields.

Morphogenetic fields, by their nature, are capable not only of (1)regeneration of lost body parts and (2) transdifferentiation ofhistological, cytochemical, ultrastructural, and molecular phenotypes,but also of (3) intrinsically recognizing and restoring parts in theirproper position. That is to say, morphogenetic fields preserve theoriginal anterior/posterior, dorsal/ventral, and right/left axes for aregenerating limb. This property, which is variously referred to asApositional information@ or sometimes Ahandedness@ is a function of theexpression of the appropriate genetic program governing a particularmorphogenetic field. (French et al., Science, 1976, 193:969-981;Wolpert, J. Theor. Biol., 1969, 23:1-47). The methods herein describedfor (1) and (2) above, representing progression of the program forselective gene activation appropriate for a particular morphogeneticfield (e.g., right forelimb) therefore inherently also accomplish (3)restoration of the structure with accurate positional information of itscomponents. For example, if a limb is amputated at the distal humerus ona right forelimb, the regenerating radius and ulna will be in theappropriate locations for a right forelimb and not a left forelimb.

Therefore, the methods described herein provide reconstitution (e.g.,limb regeneration) and, in some circumstances, creation (e.g., liverfrom pancreas remnant) of morphogenetic fields.

According to the present invention, regeneration viatransdifferentiation may be performed in situ (e.g., at the site oftrauma or injury).

Alternatively, an organ or tissue can be transdifferentiated/regeneratedin vitro, and then introduced back into the body. Thus, in one of thepreferred embodiments human pancreatic cells are regenerated andtransdifferentiated into hepatocytes by treatment with various agents incell culture, plated in a self-degrading plastic container containing aseven day supply of culture medium, and then sewn adjacent to a liverblood vessel which vascularizes the new tissue, incorporating it in theliver.

The use of in vitro transdifferentiation for regeneration of tissues andorgans insures the autologous nature of the transplant and provides agreat advantage, when employed in medical practice, by avoiding the needfor immunosuppression and decreasing the chances of transplantrejection.

In a preferred embodiment of the present invention, the method oftransdifferentiation comprises the steps of (a) destabilizing ordedifferentiating the cells, (b) transdifferentiating the destabilizedcells, and (c) stabilizing the transdifferentiated cells, causing themto differentiate.

In practicing the present invention, the tissue to betransdifferentiated/regenerated can be derived from ectoderm, mesoderm,endoderm, neural crest or extra embryonic membranes.

Destabilization/dedifferentiation may be accomplished, by, but is notlimited to, the following:

1. Administering an agent to cells at the site of injury or in culture.Agents to induce destabilization, include but are not limited to,retinoids (e.g. retinoic acid), 12-O-tetradecanoylphobol-13 acetate(TPA), 0.1 M hydrochloric acid (pH<5), hypertonic saline (saturatedNaCl), copper chelators (such as triethylenetetraminetetrahydrochloride), and heavy metals, such as copper, zinc, or cadmium.The amount of toxin administered varies from toxin to toxin but willgenerally be 1-100 μg/ml in cell culture.

2. Disintegration of the extracellular matrix (e.g. by administeringhyaluronidase or collagenase).

3. Physical separation of cells by mechanical or enzymatic methods (suchas trypsinization, EDTA treatment, or repeated needle trauma).

4. Trauma (see below).

The preferred method of destabilization depends on the accessability ofthe target tissue, the nature of the extracellular matrix structure andcomponents which hold it together. For example, when performingdestabilization of skin cells, trypsinization of the basement membraneis often the method of choice.

Trauma of any kind, including, injury caused by surgery, laser,penetration (e.g. needle), chemical, heat, visible or non-visible (e.g.UVA) light, x-radiation, infection, toxin, or immune response, tends tobe very effective in causing destabilization in many tissues. In fact,trauma is the most common natural destabilizing agent in animals thatare able to naturally regenerate organs and body parts (e.g., a predatoramputating a lizard tail or amphibian limb). Therefore, trauma can beused as a starting step in the regeneration process. Some of the modesby which trauma stimulates destabilization includes the following:

(1) Trauma of the epithelium will usually disrupt the basement membraneleading to changes in the basement membrane components such asfibronectin and laminin. These changes, in turn, are known to affectsynthesis of proteins, mRNA, and DNA (Cell Biology of ExtracellularMatrix, Hay ed., Plenum Press, 1981).

(2) Trauma may lead to changes in cells=microenvironment by affectingneighboring cells which secrete growth factors, cytokines,immunoglobulins, or other substances that affect the differentiatedstate of the cell under consideration and may cause it todedifferentiate and perhaps proliferate in the absence of normal levelsof these factors. In addition, trauma may cause cells to come intocontact with cell types or body fluids which they normally do not comeinto contact with, and this may also cause destabilization. All theseeffects on cells=microenvironment result in changes in intracellularsignaling pathways leading to changes in protein synthesis and geneexpression.

(3) Trauma, by changing the cell microenvironment, leads to changes incell shape (e.g., flattened vs. rounded) causing changes in protein andnucleic acid synthesis, (Cell Biology of Extracellular Matrix, supra)and thus affecting the cells=differentiated state. Changes in cell shapecan be dependent on such factors as the adhesive characteristics of thesurface (e.g. cell membrane, extracellular matrix, other surroundingcells).

(4) Trauma may cause the release of substances called “wound hormones”which may cause destabilization.

Some examples of how trauma may be used to stimulate destabilization ina mammal are provided in Example 18, below.

According to the present invention, following destabilization, the cellsare contacted with an effective amount of a transdifferentiation agent.Non-limiting examples of transdifferentiation agents include: guanosine,phenylthiourea or TPA.

Finally, the cells are contacted with an effective amount of astabilizing and differentiating agent. Non-limiting examples include:beta-carotene, retinoids, riboflavin, and pteridines.

All of the above-mentioned destabilizing, transdifferentiating andstabilizing reagents can be obtained commercially (e.g., from SigmaChemical, St. Louis, Mo.).

According to the present invention, effective amounts of these reagentswould broadly range between about 1 and about 100 μg/ml in the cellularmicroenvironments (e.g., cell culture medium) or between about 0.5 andabout 1,000 mg/kg body weight in a recipient animal.

It should be noted that all reagents for transdifferentiation of cellsin vitro are added directly to their culture medium. In mammalsundergoing regeneration in situ, the preferred route of administrationof the destabilization and transdifferentiation agents is topical. Whenthe organ or tissue treated is internal, direct administration isaccomplished by needle or catheter, or systemically.

The stabilizing/differentiation agents are preferably administeredsystemically, most preferably orally, enterally, by inhalation, byaerosol, rectally, etc.

In an alternative, embodiment of the invention, a transdifferentiationagent and stabilizing/differentiating agent are administeredsubstantially simultaneously instead of sequentially, e.g., guanosine isadministered together with beta-carotene. Preferably the two agents areadministered for a period of hours or days.

In another alternative embodiment of the invention, the same compound isused for the transdifferentiating step and stabilizing steps (e.g.,relatively large amounts of retinoic acid [10⁻⁴M in cell culture]). Inthis embodiment, the dedifferentiating step (e.g., amputation bymechanical trauma, blade or needle sticks) precedes the addition of thetransdifferentiation/stabilization agent.

Each of the steps in the process of transdifferentiation/regenerationtakes from hours to days to completion depending on the agent used, itsdose, method of administration and the target tissue. For example, tostimulate limb regeneration in a mammal, the hypertonic saline regimendeveloped by Rose for newts (J. Exp. Zool., 1944, 95:149-170) orrepeated sticks with a 25 gauge needle are used thrice daily for threedays after amputation.

According to the present invention, the cell destabilization phase(which can be monitored histologically [e.g., using light microscopy]and/or biochemically) is considered completed when cells have lost themorphological and biochemical characteristics which define theirphenotype. These cells start to resemble blastema cells (i.e., they havea high nucleus to cytoplasm ratio and/or lack morphologicalcharacteristics of differentiated cells). For example, in the stump ofan amputated limb, such cells are seen about two weeks after amputationin the newt Triturus viridescens.

The transdifferentiation phase is considered completed when cellsacquire some of the morphological and biochemical characteristics whichdefine their new phenotype. For example, when characteristic striatedphenotypes are seen in putative muscle cells, or axons and dendrites areseen in putative neurons.

The stabilization/differentiation phase is considered completed whencells have acquired all of their terminally differentiated morphologicaland biochemical characteristics (e.g., when one can microscopicallyand/or biochemically detect the internal storage organelles containingthe stabilization/differentiation agents, such as carotenoid droplets,deposits of riboflavin, or pteridines in pterinosomes).

In a still further embodiment, the present invention provides a methodfor identifying novel compounds that possess transdifferentiation and/orstabilization capacity. Non-limiting preferred screening systems forsaid candidate compounds are: (a) optic cup-derived retinal pigmentedepithelium (RPE) cells of the newt or mammalian eye transdifferentiatinginto lens; (b) RPE of the newt or mammalian eye transdifferentiatinginto retinal neurons; (c) mammalian pancreatic cellstransdifferentiating into hepatocytes; (d) neural crest-derived Axolotlchromatophores transdifferentiating into another pigmented cell type,and (e) mammalian terminally differentiated neural crest-derived cellstransdifferentiating into other types of neural crest derivatives (e.g.,melanocytes to neurons, cephalic connective tissue to bone, Schwanncells to neurons or bone, etc.). A preferred screening system, usingAxolotl is presented below in Example 5.

Based on the ability of some previously identified compounds to act asuniversal transdifferentiation agents in various systems (e.g.,guanosine in Axolotl chromatophore system, in newt, and in mammalian RPEsystems; copper depletion in mammalian pancreas-to-hepatocyte andmammalian RPE systems), any agent which causes transdifferentiationand/or regeneration in any of the experimental systems, will be acandidate for causing transdifferentiation and regeneration in othertissues and in other animals. In addition, agents that cause cells(e.g., neural crest cells) to differentiate along a particular pathwayduring embryogenesis will be candidates for transdifferentiation agentsin adults.

Thus, in one embodiment Stage I candidate compounds will be tested fortheir ability to convert mature adult human skin melanocytes into neuralstem cells. A dose response curve will be generated by concurrentlyincubating, in separate vials, cultured cells with a test substance atconcentrations ranging between about 10⁻³ μg/ml and about 100 μg/ml, theconcentration increasing by half log units. Cells will be incubated withthe test compounds from one to 21 days. Cells will be examined forevidence of transdifferentiation by light or electron microscopy,biochemical tests, and immunological labeling with the criteria oftransdifferentiation depending on the stem cells used and the new tissuetype desired. For example, the transdifferentiation of melanocytes toneural crest stem cells, will be observed as a loss of melanosomes andmelanin.

In a still further embodiment, Stage II candidate compounds will betested for their ability to convert the cells resulting from Stage Iinto terminally differentiated phenotypes, such as neurons, Schwanncells, cartilage cells, and fibroblasts. A dose response curve will begenerated by concurrently incubating, in separate vials, cultured cellswhich have been previously transdifferentiated, with a second testsubstance at concentrations ranging between about 10⁻³ μg/ml and about100 μg/ml, the concentration increasing by half log units. Cells will beincubated with the test compounds from one to 21 days. Cells will beexamined for evidence of redifferentiation by light or electronmicroscopy, biochemical tests, and immunological labeling with thecriteria of transdifferentiation depending on the stem cells used andthe new tissue type desired.

Examples of chemicals which can be screened by the above methodsinclude: purines and pyrimidines, nucleosides and their derivatives,retinoids, carotenoids, laminin, fibronectin, growth factors, cytokines,ommochromes, thioureas, chelating agents, and metals (such as zinc,copper, cadmium). Depletion or supplementation of substances normallypresent in culture media can also be screened by the above method (Brentet al., Am. J. Pathol., 1999, 137:1121-1142).

Based on the above-mentioned experimental evidence from varioustransdifferentiation systems, low toxicity and good solubilityproperties, guanosine is a particularly preferred compound for use as atransdifferentiation agent in the present invention. Thus, as shownbelow in Example 1, in gerbils oral administration of guanosine (atransdifferentiation agent) followed by beta-carotene (a stabilizingagent) after surgical removal of a lens, led to regeneration of the lensfrom RPE. It should be noted that in this case, thedestabilization/dedifferentiation agent was the trauma caused by thesurgery used to remove the lens.

In further alternative embodiments guanosine may be substituted for withguanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), adenosine, cytosine, thymidine, uridine and theirphosphates, or catecholamines (such as norepinephrine, and epinephrine).

In light of the above-mentioned experimental evidence, copper is anotherpreferred compound for use as a transdifferentiation agent in thepresent invention. Agents which affect copper levels in the body (and inspecific tissues) thus may also have activity as transdifferentiationagents. Some of the substances that cause changes in copper levelsinclude the following:

(I) Metals such as zinc, cadmium and iron (Linder and Hazegh-Azam, Am J.Clin Nutr, 1996, 63:797S-811S);

(II) Copper chelators such as trien (Rao and Reddy, supra),penicillamine (Brewer, Copper Transport and Its Disorders, Leone andMercer eds., Plenum Publishers, 1999);

(III) Agents that affect the absorption of copper such as zinc acetate(Brewer, supra);

(IV) Enzymes that affect the metabolism of copper and/or use copper astheir cofactor (e.g., tyrosinase, superoxide dismutase, hyaluronidase);copper binding proteins (e.g., ceruloplasmin or metallothiein) (Linderand Hazegh-Azam, supra);

(V) Substances that affect the synthesis or degradation of copperbinding proteins/enzymes (e.g., retinoic acid [Song and Levenson, Int.J. Vitam. Nutr. Res., 1997, 67:141-144]);

(VI) Copper binding non-proteinaceous compounds (e.g., ascorbate [Itohand Eguchi, Dev. Biol., 1986, 115:353-362; Droudin et al., Free RadicBiol Med, 1996, 21:261-273], various thioureas [Masuda and Eguchi, CellStructure and Function, 1984, 9:25-35], guanosine, adenosine [Masuda andEguchi., Inorganic Chemistry, 1990, 29:3631], cytosine [Palaniandavar etal., J. Chemical Soc., 1996, 7:1333] and their phosphates).

Copper deficient diets (Yoshida et al., J. Neurooncol., 1993, 17:91-97)and copper depleted media (Percival and Layden-Patrice, J. Nutr., 1992,122:2424-2429) for cells are other methods for changing copper levels.

One way by which the dedifferentiating and transdifferentiating effectof copper is believed to be exerted is by its acting as a cofactor ofenzymes which generate products characteristic of the terminallydifferentiated cells (e.g., tyrosinase that leads to production ofmelanin). Copper is also thought to cause transdifferentiation byaffecting the amount of free guanosine (Tu and Friederich, Biochem.,1968, 7:4367-4372; Maskos, Acta Biochemica Polonica, 1978, 2:101-111)and by direct binding to DNA and RNA (Iyengar, Acta Anat., 1983,115:357-360; Wong et al., Can J. Biochem., 1974, 52:950-958; Sorokin etal., J. Inorg. Biochem., 1996, 63:79-98) which leads to their break-down(i.e., stand scission and fragmentation; Yamafuji et al., Enzymologia,1971, 40:107-119; Dowjat et al., BioMetals, 1996, 9:327-335). Finally,copper is angiogenic (Brem et al., Am. J. Pathol., 1990, 137:1121-1142;Yoshida et al., Neurosurgery, 1995, 37:287-293) and thus supplementationof copper encourages growth of tissues and differentiation. It can betherefore concluded that other substances which act in a manner similarto copper would be candidate transdifferentiation agents.

Another group of preferred compounds for use as transdifferentiationagents according to the present invention includes substances involvedin biochemical pathways producing pigments (such as melanin,ommochromes, hemocyanin, carotenoids, flavonoids, and pteridines).According to the present invention, the preferred experimental system totest these compounds is either Axolotl chromatophore system, or Kupfferstem cell system in which stem cells differentiate into the full line ofneural crest derivatives (Nozue, Acta Anat., 1990, 107:188-197; Labat etal., Biomed Pharmacother., 2000, 54:146-162).

Novel dedifferentiating, transdifferentiating and stabilizing compoundsidentified according to the method disclosed herein may be furthertested for their effectiveness and pharmaceutical acceptability.Preferred agents will meet the following criteria:

1. The ability to produce or regenerate tissues/organs from a repeatedtoxic or traumatic injury which function most like the original (e.g.,regeneration of a lens in a gerbil which is most like the original intransparency, geometric shape, ability to focus light, accommodate,etc.);

2. Minimal local and systemic toxicity (e.g., guanosine andbeta-carotene);

3. The ability to act rapidly and in relatively small amounts;

4. The ability to be made into easily administered pharmaceuticalpreparations.

Without wishing to be bound by theory, it is believed that theregenerative ability of animals is critically dependent on their diet.Organisms which ingest food that includes large quantities of purines,pteridines and carotenoids, or precursors of each of these compounds,maintain multi-potent cells which may more easily undergotransdifferentiation. The effect of diet on transdifferentiation isexemplified by the loss of regenerative ability as amphibians maturefrom larvae (herbivorous diet) to adult (carnivorous diet), and bygreatly decreased ability of vertebrates which have a diet deficient incarotenoid containing foods, to regenerate lost body parts.

Thus, according to the present invention, the optimal conditions forregeneration in vertebrates, and specifically in mammals include:

6. A diet supplemented for a month prior to injury with beta-carotene(1000 mg/kg), which includes the 9 cis isomer as well as othercarotenoids such as lutein, and zeaxanthin; under these conditionsoptimal regeneration will be observed in mammals that are high caroteneaccumulators (such as gerbils and humans); human patients who consumelarge amounts of carotenoid-containing vegetables are particularly goodcandidates for the methods disclosed herein;

7. Preparing the site of regeneration by local topical application tothe wound surface of a saturated HCl solution to facilitate electricalproperties (e.g., by immersion of the digit stump into hypertonic salinefor 1 hour, twice per day):

8. A photo period of 8-16 hours light per 24 hours depending on thetissue and species, with a source of natural light which includes the UVspectrum during the redifferentiation step.

When employing conditions listed above, the optimal regeneration will beobserved in small structures which have a high density ofchromatophores, such as infant=s fingers or toes accidentally amputatedat any level including the metacarpal-phalangeal joint.

In order to produce a particular cell type, it is necessary to select aparticular agent, close and exposure time for each of the agents duringthe following phases: destabilization, dedifferentiation,transdifferentiation, stabilization.

To select the appropriate treatment, a matrix of effects is created bysystematically varying each factor above. For example, if it is desiredto produce a particular type of spinal motor neuron using retinoic acidas the agent for transdifferentiation. A matrix will be set up whereinretinoic acid is tested at different half log incrementalconcentrations, e.g., ranging from 10⁻⁹ molar to 10⁻⁴ molar, with otherfactors being held constant. For each of these culture tubes, the cellsresulting from the stabilization phase will be tested histochemicallyfor the characteristics of the spinal motor around. Once a particularconcentration of retinoic acid is selected in this manner, the processwill be fine-tuned by setting up a matrix of exposure times which aresystematically incremented e.g. exposure times of doses from one day toseven days in one day increments. Agents during the destabilization andstabilization phases will similarly be varied to choose the exactlycorrect agent, it's dose and exposure times to result in a particularcell type.

The starting point to use for the range of dose can be determined asfollows: if the agent has ever been reported used in cell culture ofsome type, the starting dose will be about the dose reported in theliterature. If the agent has not been used in the cell culture, thisstarting dose will be that of any similar chemicals reported in theliterature used in cell culture. If the agent has never been reported, asystematically incremented range, either in molar concentrations or byweight is chosen. If the cells at a particular dose die, then the doseis too high.

If the cells at a particular dose show no effect, then the dose is toolow, and by bracketing results in this matter and iterating theprocedure, the proper dose will be selected.

In those cases where the agent is not a chemical, that agent will stillbe systematically varied during the testing process. For example, if theagent is trauma created by the use of a scalpel, the size of theincision will be systematically varied in a matrix, other factors heldconstant, and the resulting cell types identified and classifiedhistochemically or immunocytochemically.

In another embodiment, the present invention provides a method forscreening anti-cancer agents by testing their ability to inducetransdifferentiation of cancer cells (e.g., melanoma cells) into benignterminally differentiated cells. In a preferred embodiment, the leasttoxic and the most efficient transdifferentiating agent is firstselected using in vitro cultured cancer cells derived from a particularpatient. The selected agent is then administered to patient, and theregression of cancer is monitored by standard techniques. In this way anoptimum individualized anti-cancer treatment is achieved.

In a still further embodiment, the present invention provides a methodfor inhibiting the progression of an antibody-dependent autoimmunedisease (such as lupus or myasthenia gravis). Said method involveslowering the titer of autoreactive antibodies present in the blood of apatient suffering from an autoimmune disease by regular dialysis of theblood through the matrix containing immobilized patient=s cells reactivewith these antibodies. According to the present invention, the unlimitedsupply of such immobilized cells could be obtained by in vitrotransdifferentiation of cultured cells derived from the same patient.Because these are the patient=s own cells, no new unwanted antibodieswould be generated, and the adverse effects would be minimized.

The present invention is described below in working examples which areintended to further illustrate the invention without limiting the scopethereof.

Example 1 Stimulating the Regeneration of a Lens in a Gerbil Materialsand Methods

The effect of guanosine and beta-carotene on stimulating theregeneration of an amputated lens was examined in Mongolian Gerbils(Meriones unguiculatus).

Animals: 4 week-old Mongolian Gerbils from Charles River LaboratoriesWilmington, Mass.

General Conditions:

Temperature:

Days 1-23: 22° C.

Day 24: 26° C.

Day 25: 29° C.

Day 26: 32° C.

Days 27-61: 35° C.

Photoperiod: 12 hrs light; 12 hrs dark

TABLE I Gerbil Protocol Groups Group No. Diet Days 1-33 Diet Days 34-62Males Females 101 A* A 4 4 102 A, CT A 5 5 103 A, CT, G A 5 5 104 A, CT,B A, B 5 5 105 A, CT, G, B A, B 5 5 106 A, G A 4 4 107 A, G, B A, B 4 4108 A, B A, B 4 4 Totals 36 36 *A = AIN 93G (a standard rodent dietavailable from Dyets, Inc., Bethlehem, PA), C = copper deficient dietwith 30% of normal copper (normal copper = 6 mg/kg diet: copperdeficient = 30% normal = 1.8 mg copper per kg diet), T = 0.5%Triethylenetetramine Tetrahydrochloride (trien), B = 1% DunaliellaBeta-carotene with two times the usual amount of vitamin K (normal Vit.K = 0.75 mg Vit. K per kg diet; double Vit. K = 1.5 mg per kg diet), G =1% Guanosine.

Diets (Manufactured by Dyets, Inc., Bethlehem, Pa.):

Based on usual food intakes, it is estimated that animals took in theapproximate amounts of chemicals as follows. For diets containingguanosine, 1000 mg/kg/day Guanosine was consumed by each animal. Fordiets containing beta-carotene, 1000 mg/kg/day Beta-carotene wasconsumed by each animal.

Animal Laboratory Location: PSL, East Brunswick, N.J. HistologicalLaboratory: Colorado Histo-Prep, Inc., Fort Collins, Colo.

The experimental animals were fed diets disclosed above to enhancededifferentiation of the iris epithelium, and to subsequently lead totransdifferentiation of the iris epithelium into lens. A control groupfed a standard AlN diet was included. Throughout the experiment, theanimals were examined daily by a licensed veterinarian.

The temperature was gradually raised by 3° C. per day for four daysprior to surgery to reach a temperature of 35° C. which at the time wasbelieved to help lens regeneration. One animal from each group wassacrificed on clay 27 and preserved in formalin.

The following surgical protocol was followed for each remaining animalon day 28:

1. A drop of 1% tropicamide (Bausch & Lomb, Tampa, Fla.) was placed inthe right eye;

2. The animal was anesthetized by IP injection of ketamine withxylazine;

3. A diamond knife (Accutome, Malvern, Pa.) was used to make a cut inthe cornea of the right eye, which was expanded with a Vanas scissors(Miltex, Bethpage, N.Y.), so that an incision at the limbus of about180-270° was made;

9. The lens was lifted out with a Tyrell Loop (Miltex, Bethpage, N.Y.)when gentle pressure was exerted superiorly on the eyeball by a glovedfinger;

10. The removed lenses were retained in formalin.

After surgery, the animal diet was changed to the standard AlN diet,except that the group which had received AGB continued to receivebeta-carotene via the AB diet. Some pellets were put in the cage so thatafter surgery, the animal would not have to reach up to the lid of thewire cage to get the food.

The animal eyes were examined with a magnifying glass without anesthesiaweekly. However, since this animal has a dark eye, little detail couldbe ascertained.

On day 61, the animals were anesthetized by CO₂ and decapitated. Theheads were placed in Davidson=s fixative for 18 hours and then switchedto neutral buffered formalin. The bodies were fixed in neutral bufferedformalin.

All operated eyes fixed on day 61 were photographed. All heads from the50 animals that survived to the end of the experiment were sent toColorado Histoprep. The operated eye was dissected and serial 5μsagittal sections up to the approximately mid-saggital plane were cut ona rotary microtome. For all specimens, at least 50 5μ serial sectionsfrom the mid-saggital area were prepared. The slides were processedroutinely for histology and stained with Hematoxylin and Eosin.

Results and Conclusion

The operated eyes were flat after surgery. It was noticed that someanimals=eyes remained partially or completely closed over the nextmonth. However, some animals=eyes gradually became less flat and someoperated to protrude almost normally.

Remarkably, about 40% of the experimental eyes showed lenses which hadclearly regenerated. These often showed a stalk connecting them to theiris pigment epithelium (as seen in newt lens regeneration), as well asindividual lens cells which clearly contained melanosomes, identifyingthem as having derived from the pigmented epithelium. These lensesextended over many histological sections, showed well formed lensescontaining organized lens cells in various stages of forming fibers, asis seen embryologically. They generally were without cystic or necroticmaterial. When the extracted formalin-fixed whole lens was examined andcompared to the histology of the regenerated lens for each animal, itwas clear that in practically all cases, almost the entire lens had beenremoved, with the possible exception of a small amount of lensepithelium, and it was not likely that the regenerated lens was due toretained material which had proliferated. The stalk to the iris pigmentepithelium, as well as individual cells containing melanosomes, showingderivation from pigment epithelium, confirmed regeneration.

Some of the operated eyes showed no lens material whatsoever (seebelow). Others showed lens material which had clearly been retainedafter the surgery. (It is well known from human cataract surgery as wellas experimental models that it is often impossible to remove the entirelens so that not even a few cells of the lens epithelium remain. This isproblematic clinically, since retained lens epithelium is known toproliferate and complicate the lens implants=function). Retained lensmaterial could be identified histologically because it was generallynecrotic, cystic, showed mature lens fibers, had no connection to theiris pigment epithelium, only extended over a few serial sections and byother histologic criteria. Microscopic examination and photographs ofthe formalin fixed extracted lenses showed that although most wereremoved grossly intact, all had at least small epithelial defectssuggesting that some epithelial tissue had been left behind.

TABLE 2 Histological Observations on Surviving Animals Group Diet DaysDiet Days No Retained Regenerated Group Percent No. 1-32 33-62 Lens LensLens Total Regenerated 101 A A 2 4 0 6 0% 102 A-CT A 1 4 3 8 38% 103A-CT-G A 1 4 0 5 0% 104 A-CT-B A-B 3 2 3 8 38% 105 A-CT-G-B A-B 1 2 4 765% 106 A-G A 3 0 3 6 50% 107 A-G-B A-B 4 0 3 7 43% 108 A-B A-B 1 0 2 366%

As can be seen from the above table, none of the 6 surviving controlanimals (Group 101) showed regenerated lenses, although 4 showed smallretained epithelium which had proliferated.

18 of the total 44 surviving experimental animals (Groups 102-108)showed regenerated lenses (41%).

There were clear differences among the groups in the quality of theregenerated lenses, with generally the best (largest, most normalappearing in overall architecture and cytology) were from group 106(initial guanosine diet) and group 107 (initial guanosine andbeta-carotene diet).

Example 2 Enhanced Protocols for Stimulating the Regeneration of a Lensin a Gerbil

1. An Improved Protocol for Stimulating the Regeneration of a Lens in aGerbil

The following experiment on lens regeneration in a gerbil will beconducted to improve the experimental protocol for all further studies,and to obtain additional specimens for histological and ultrastructuralanalysis.

Thirty six 3-week-old Mongolian Gerbils will be obtained from CharlesRiver Laboratories (Wilmington, Mass.) and acclimated for one week atProduct Safety Laboratories (East Brunswick. N.J.) animal carefacilities. The temperature will be kept at 22° C. throughout theexperiment because it is now believed that the higher temperatures (35°C.) may be unnecessary. They will be kept on a 12 hour light/darkphotoperiod and fed Purina Rat Chow during the one week acclimationperiod. Three groups of 12 animals each will be used.

GROUPS DIET 1 DIET 2 (1) Zinc Acetate 0.1% AIN (2) Guanosine 1% +Beta-carotene 1% Beta-carotene 1% (3) Control AIN diet AIN

The rationale for the use of zinc acetate rather than the previous 30%copper diet with trien is as follows. Zinc acetate is available as aprescription drug (Galzin, Lemmon Co., Sellersville, Pa.). Moreover, ithas little adverse effects and thus the mortality seen from the copperdeficiency diet with trien should be avoided. The dose of zinc acetatewill be chosen based on published studies on its use in rodents to lowersystemic copper (Approved Package for NDA 02458 Galzin Capsules, 1997,FDA, Rockville, Md.).

The animals will receive Diet 1 for 2 weeks to promote dedifferentiationof the RPE. The animals will be anesthetized using methoxyflurane, thesafest and overall most preferable anesthetic in gerbils (Norris, Lab.Anim., 1981, 15:153-155). Lentectomy will be performed using theprocedures described in Example 1. The animals will receive Diet 2 for 6weeks. This is 2 weeks longer than the previous protocol and shouldpermit completion of the lens regeneration process documented above.

At the end of Diet 1, two animals for electron microscopy (EM) and twofor histology will be sacrificed from each group. At end of Diet 2, fouranimals for EM and four for histology will be sacrificed from eachgroup.

Fixation methods will be as previously described.

Sacrificed animals will be decapitated. The specimens will be sent to ahistology laboratory where the eyes will be dissected out. This ispreferable to immediately dissecting out the eyes as it will minimizehandling the eyes until fixed and will help histologists to properlyorient the eye on the block.

2. Adjustment of the Dose and the Mode of Administration of ActiveCompounds Used in the Lens Regeneration

48 3-week old Mongolian Gerbils will be obtained from Charles RiverLaboratories and divided into the following groups of 6 animals each.

Group Regimen 1 Control AIN diet 2 Guanosine 0.05% in diet 3 Guanosine0.10% in diet 4 Guanosine 0.50% in diet 5 Guanosine 1.00% in diet 6Guanosine AIN diet; saturated solution of guanosine injected afterlentectomy 7 Guanosine AIN diet; half-saturated solution of guanosineinjected after lentectomy 8 Guanosine AIN diet; crystal of guanosineinserted after lentectomy

Groups 1-5 will enable the investigator to determine whether a muchsmaller dose than was previously used will be adequate to stimulate lensregeneration. Experimental groups 6-8 will allow to determine whethersimply instilling guanosine into the lentectomized eye, rather thanproviding it in the diet will be adequate to stimulate lensregeneration. These conditions are similar to published regimens forguanidine derivative-mediated transdifferentiation of ventral iris tolens in the newt (Eguchi and Watanabe. J. Embryol. Exp. Morphol., 1973,30:63-71). The remainder of the protocol (i.e., photoperiod, length ofDiet 1, lentectomy, etc.) will be the same as described above (part 1).Diet 2 will always be AlN for this experiment.

3. Standardization of the Lens Regeneration Experiment

The purpose of this experiment is to create a standardized series ofstages for future studies.

98 animals will be obtained from Charles River Laboratories and dividedinto two equal groups.

GROUP DIET 1 DIET 2 (1) Control (AIN) AIN (2) Guanosine 1% +Beta-carotene 1% Beta-carotene 1%

The animals are fed Diet 1 for 2 weeks and then Diet 2 for 6 weeks.During Diet 1, every 3 days, three animals from each group aresacrificed. During the first 4 weeks of Diet 2, every 3 days, threeanimals from each group are sacrificed.

During the last 2 weeks of Diet 2, every week, three animals from eachgroup are sacrificed. Other conditions are as described above. Specimensare prepared for histological analysis as previously described.

Example 3 Lens Regeneration in Mice and Rats

24 three-week-old strain of REJ mice from Jackson Labs will be obtained.They will be divided into two equal groups.

GROUP DIET 1 DIET 2 (1) Guanosine 1% + Beta-carotene 1% Beta-carotene 1%(2) Control AIN diet AIN

The temperature will be maintained at 22° C. throughout the experiment.The photoperiod will be 1.2 hours light/dark. The animals will be fedDiet 1 for 2 weeks. Then the animals will be anesthetized with ketamineand xylazine, and then lentectomy will be performed as previouslydescribed for gerbils. Then the animals will be fed Diet 2 for 6 weeks.At the end of Diet 1, two animals for EM and two for histology will besacrificed from each group. At end of Diet 2 four animals for EM andfour for histology will be sacrificed.

The same protocol will be repeated with Sprague Dawley rats.

Example 4 Stimulating Retinal Regeneration in a Mammal (Gerbil)

Newts and other amphibians are able to regenerate neural retina fromretinal pigmented epithelium (RPE) after trauma or experimental retinaldetachment (Reyer, pp. 309-390, In: Handbook of Sensory Physiology VII,Crescitelli ed., Springer-Verlag, 1977). Even some amphibians which areunable to regenerate lens are able to regenerate retina (Reyer, supra).It is believed that the same protocol which has permitted lensregeneration will also permit retinal (neural retina, photoreceptor)regeneration.

68 five-week-old Mongolian Gerbils will be obtained from Charles RiverLaboratories and divided into two equal groups.

GROUP DIET 1 DIET 2 (1) Guanosine 1% + Beta-carotene 1% (2) Control AINdiet Control AIN diet

The preferred source of beta-carotene will be the algae Dunalielaavailable commercially, e.g., Henkel Corp., La Grange, Ill. As inprevious protocols. Diet 1 will be used for 19 days and Diet 2 for 6weeks. On day 15, experimental retinal detachments will be made in theright eyes using techniques published for amphibians (e.g., Hasegawa,Embryologia, 1958, 4:1-32; Stone, J. Exp. Zool., 1950, 113:9-32; Keefe,J. Exp. Zool., 1973, 184:185-206) or mammals (e.g. Mervin et al., Am J.Ophthalmol., 1999, 128:155-164; Chan et al., Retina, 1996, 16:139-44;Takeuchi et al., Invest. Ophthalmol. Vis. Sci., 1995, 36:1298-305), orvariations of these techniques.

Four animals will be sacrificed 1 day after the detachment is created,and then four animals per week for five weeks. At the end of six weeksall remaining animals will be sacrificed. The right eyes will beprepared for histological analysis.

Example 5 Axolotl Assay System for Transdifferentiation Agents

A series of transdifferentiation experiments were conducted on Axolotls.These experiments demonstrated the utility of this method as an assayfor discovering and comparing transdifferentiation and/or stabilizingagents.

The evidence presented herein showing that copper depletion acts as atransdifferentiation agent in this amphibian skin assay system (as ithas conclusively been demonstrated to be a transdifferentiation agent inthe rat pancreas to hepatocyte transdifferentiation system) reinforcesthe statements expressed previously, that (1) transdifferentiationagents tend to be universal in vertebrates, and (2) agents identified inone assay system are likely to work in all vertebrates and in severaldifferent organ systems.

This assay method, as described below, has led to the identification ofGMP (guanosine 5′-monophosphate), cGMP (guanosine 3′,5′-cyclicmonophosphate), as well as zinc acetate and inositol astransdifferentiation agents.

Larval Axolotls have an epidermis on a thin basement membrane, underwhich is a thick collagen layer (green when stained with Gomoritrichrome for light microscopy.) Only 3 types of cells are presentimmediately below and slightly embeded in the collagen layer—(a)fibroblasts, which are generally fusiform and have a characteristiclight and ultrastructural appearance, (b) melanophores, which containnumerous round or oval black melanosomes, as well as somepremelanosomes, and (c) xanthophores, which, contain organelles known aspterinosomes. These appear as vacuoles in light microscopy and vaculeswith wispy or concenteric layered material in electron microscopy. Ingeneral, melanosomes are normally found only in melanophores andpterinosomes in xanthophores. Evidence presented by Frost, and reviewedby Bagnara, showed that guanosine acted as a transdifferentiation agentwhen fed to larval axolotls, causing melanophores to convert toxanthophores as shown by ultrastructural studies.

All Axolotls were 3-5 cm in length at the beginning of the experimentand were obtained from the Indiana University Axolotl Colony,Bloomington, Ind.). They were maintained on beef liver (example A below)or fish pellets (Indiana University Axolotl Colony, Bloomington, Ind.)(examples B and C below).

Temperature was 22° C. with a 12 hour light/12 hour dark

photoperiod. Water was changed daily before feeding, and when theanimals were removed from feeding an hour later. They were fed aboutevery day. Every other day, the experimental substance was sprinkled onthe beef or fish pellets. All experimental reagents were obtained fromSigma (St. Louis Mo.). In example A, the animal was kept in a 0.75 quartstainless steel dish with a bottom raised to a plateau in the middle.For feeding, beef liver was placed on the raised bottom partially in andpartially out of the water. This permitted the animal to sense the foodin the water but prevented the cGMP sprinkled on the beef from washingaway. For examples B and

C, the animals were kept in individual 280 ml plastic containers thatwere replaced twice per week. For feeding the animals were transferredto smaller plastic containers (weighing canoes) which have one sidesloped. The pellets were placed on the slope, partially in and partiallyout of the water. This permitted the animal to sense the food in thewater but prevented the cGMP sprinkled on the pellet from washing away.Deer Park Spring Water (Breingsville, Pa.) was used exclusively.

The animals were observed about weekly under an Olympus SZstereomicroscope and most were photographed prior to sacrifice orshortly (hours) after death. Animals were fixed in 10% Formalin and 4 mmpunch biopsies of skin were processed routinely for histology (GomoriTrichrome) by Charles River Laboratories (Wilmington, Mass.). Slideswere examined in an Olympus (Woodbury, N.Y.) BX40 microscope.

Three animals (a control and two GMP fed animals) were biopsied with a 2mm punch and fixed in Trump=s fixative for 24 hours, and processedroutinely for electron microscopy. They were postfixed in 2% osmiumtetroxide, dehydrated in a seriels of alcohols and embedded inEpon-Araldite (1:1). Processing and photography were performed at theElectron Imaging Facility, Rutger=s University, Piscataway, N.J.

Example A

An Axolotl was fed cGMP by the above method for 22 days and then died.Examination of Gomori trichrome stained skin showed presence ofmelanosomes and pterinosomes in several cells. This was evidence of thetransdifferentiation caused by cGMP of melanophores to xanthophores. Theanimal was also lighter by inspection. A control animal sacrificed abouta week later did not show any cells with both organelles.

Example B

Two Axolotls were fed GMP as described above for 67 clays. Electronmicroscopic examination of one animal=s skin showed many cells in whichboth melanosomes and pterinosomes were present (FIG. 1). Some cellsshowed organelles which appeared to be hybrids of melanosomes andpterinosomes (e.g. the irregular vacuolar structure was similar to thatof pterinosomes but there were dark material such as seen in melanosomes(FIGS. 2 and 3). It is believed that such organelles demonstratetransdifferentiation at the organelle level. The second animal showedclear evidence of transdifferentiation by the presence of cellscontaining both pterinosomes and melanosomes in Gomori trichrome stainedlight sections (FIG. 4). These animals were also markedly lighter byinspection and had a yellow hue. A control animal also sacrificed on day67 was in general without cells which had hybrid organelles or cellscontaining both types of organelles.

Example C

Two animals were fed Zinc Acetate as described above. One animal diedafter 7 days and one animal died after 12 days. A control animal wassacrificed a week after the second zinc fed animal died. Gomoritrichrome stained slides revealed cells with both melanosomes andpterinosomes, thus providing evidence that zinc acetate was causingtransdifferentiation of melanophores into xanthophores. The controlanimal was in general without cells which had hybrid organelles or cellscontaining both types of organelles.

Similar experiments have been conducted in which the animals have beenfed myo-inositol, trien (triethylenetetramine tetrahydrochloride),guanosine, and cytosine. Based on macro observations and photographs ofthe whole animals, it appears that guanosine (as reported previously byFrost), trien and myo-inositol are agents which transdifferentiatemelanophores to xanthophores, while cytosine transdifferentiatesxanthophores to melanophores.

Example 6 Restoration of Sight in a Dog Blinded by Retinal Degenerationby Stimulation of Retinal Regeneration

A dog (male Miniature Schnauzer, 4 calendar years old, weight about 10kg) was noticed by the owner to be frequently bumping into objects. Anexperienced veterinary ophthalmologist made the diagnosis of SuddenAcquired Retinal Degeneration (O=Toole et al., Veterinary Record, 1992,130:157-161; Miller et al., J. Vet Res., 1998, 59:149-152) after makingthe following observations on examination: The dog acted completelyblind. The pupils were completely dilated. There was no indication ofglaucoma, injury or metabolic disease. The dog was started on guanosine(Sigma; St. Louis, Mo.) ½ teaspoon per day (2.5 gm/day; about 250 mg/kgbody weight/day) and Betatene (Henkel, 7.5% Dunaliella beta-carotene) ½teaspoon per day (2.5 gm/day; about 18.75 mg/kg body weight/day).

Two weeks later examination revealed the dog to be still clinicallyblind, but the pupils constricted slightly when stimulated with brightfocal illumination. The guanosine and Betatene were continued.

Four weeks later the dog was examined again. Pupillary reflexes weresimilar to those noted in the previous examination. When theophthalmologist dropped pieces of cotton a few feet in front of the dog,he followed them most of the time as they dropped to the floor. Sincethe cotton could not be heard, smelled, or felt, it was apparent thatthe dog could see it.

The cause of SARD is not known, and no predictably successful treatmenthas been published. Histologically, there is rapid loss of rod and coneouter segments followed by a degeneration of the retinal layers.Different zones of the retina are not equally affected, in contrast tomany of the hereditary canine retinal degenerations that have appearedin the literature.

Because of the almost hopeless prognosis of SARD, and the apparentimprovement in this dog's vision, the veterinary ophthalmologistconcluded that the transdifferentiation protocol described herein abovewas responsible for demonstrable improvement in vision in this dog. Inthis example the degeneration of the retina (whatever the ultimateetiology of SARD) served as the destabilizing (dedifferentiating) agent.Guanosine served as the transdifferentiation agent of RPE into neuralretina, and beta-carotene (as Betatene 7.5%) served as the stabilizingagent.

Thus, the methods described led to transdifferentiation of retinalpigment epithelium into neural retina, regenerating the retina, andrestoring to a measurable degree, vision to a dog blinded by retinaldegeneration.

The dog was examined again 4 weeks later. The owner believed that hecould definitely see better in poor lighting than bright lighting. Theveterinary ophthalmologist examined him and agreed. When he droppedpieces of cotton in front of the dog, the clog followed the cotton mostof time, he did not follow it as frequently in standard exam roomlighting as he did in poor lighting.

The veterinary ophthalmologist suggested that the dog continue the oralbeta-carotene, but at a higher dose. The veterinary ophthalmologistconcluded that the visual function was visibly better than in theprevious examination.

Example 7 Regeneration of Islet Cells in the Human Pancreas

Islet cells from the human pancreas will be regenerated. A portion ofthe human pancreas (10 g or more) from an individual will be excised.Cells will be cultured in vitro using culture methods similar to thoseof Githens et al. (In Vitro Cell. Dev. Biol., 1994, 30A:622-635). Thecells will then be destabilized by the addition of retinoic acid (1-10μg/ml). Trans-differentiation will be accomplished by the addition ofguanosine (1-100 μg/ml). A carotenoid will then be administered as astabilization agent (1-30 mg/ml in tetrahydrofuran). The resulting isletcells will be proliferated in culture. The islet cells will then bere-implanted into the patients body by injection into the bloodstream orpancreatic blood vessel. The autologous nature of the islet cells willavoid the need for immunosuppression. Preferentially, infants orchildren under the age of 5, without concurrent diseases will besubjects for this procedure.

Example 8 Regeneration of Human Liver Hepatocytes

Hepatocytes will be regenerated from the human pancreas. A portion ofthe human liver (e.g., 1-100 g) from an individual will be excised.Cells will be cultured in vitro. The cells will then be destabilized bythe addition of retinoic acid (1-100 μg/ml). Transdifferentiation willbe accomplished by the addition of guanosine (1-100 μg/ml). A carotenoidwill then be administered as a stabilization agent (beta-carotene, 1-100mg/ml). The resulting hepatocytes will be proliferated in culture. Thehepatocytes will then be re-implanted into the patients body. Theautologous nature of the hepatocytes will avoid the need forimmunosuppression. Preferentially, infants or children under the age of5, without concurrent diseases will be subjects for this procedure.

Example 9

Kupffer stem cells of the liver and other macrophages in organs will becultured in vitro and treated with various agents promoting theirtransdifferentiation into other cells types such as melanocytes, bone,connective tissue, neurons (Sichel et al., Pigm. Cell Res., 1997,10:271-289; Labat et al., 2000, supra).

Example 10 Transdifferentiation of Neural Crest-Derived Cells

Terminally differentiated cells derived from the neural crest will beinduced to transdifferentiate into stem cells or other neuralcrest-derived terminally differentiated cells by a one- or two-stepprocess. Preferred examples include transdifferentiation of melanocytesor pineal cells to neurons, bone or muscle; cephalic connective tissueto bone, Schwann cells to neurons or bone, etc.

The cells will first be contacted in vitro with a transdifferentiatingagent such as guanosine, phenylthiourea, or TPA (1-100 μg/ml). The cellswill then be contacted with a differentiating agent such asbeta-carotene, retinoids, riboflavin or pteridines (0.1-100 μg/ml).

1. Treatment of Parkinson=s Disease by Transdifferentiation ofMelanocytes into Neurons

Patients suffering from Parkinson=s Disease will be treated with brainimplants df neural cells transdifferentiated in vitro from a patients=own melanocytes. Melanocytes will be harvested and cultured from abiopsy of adult human skin taken from a patient=s back. Cells will bededifferentiated by incubation for one week in culture medium containingcyclic guanosine monophosphate (cGMP) at a concentration of 20 μg/ml. Atthe end of a week the cells will show evidence of de-pigmentationincluding loss of melanosomes and melanin. The cells will betransdifferentiated and stabilized by incubation in a medium containingbasic fibroblast growth factor (30 ng/ml; Sigma. St. Louis, Mo.). Aftera week, the appearance of neural and glial cells in culture will beconfirmed microscopically and/or biochemically and/or immunologically.The neural cells will be then harvested and used for autologous brainimplants in Parkinson's Disease patients, using methods documented forembryonic cell implants (Brundin et al., Brain, 2000, 123:1380-1390).

2. Treatment of Cataracts by Transdifferentiation of Melanocytes intoLens

Patients suffering from cataracts will have lens cells replaced usingtransdifferentiated cells grown in culture. Melanocytes will beharvested and cultured from a biopsy of adult human skin taken from apatient=s back. Cells will be dedifferentiated by incubation for oneweek in copper-depleted culture medium. The cells will be thentransdifferentiated into lens cells by incubation for one week in amedia containing ascorbic acid 0.2 mM. The appearance of lens cells inculture will be identified by immunological staining for crystallins.The new lens cells will be harvested and used for autologous implants inpatients requiring an intraocular lens due to a cataract.

3. Treatment of a Knee Injury by Transdifferentiation of Melanocytesinto Cartilage Cells

Patients suffering from a knee cartilage injury will have tissuereplaced using transdifferentiated cells grown in culture. Melanocyteswill be harvested and cultured from a skin biopsy taken from a patient=sarm. Cells will be first dedifferentiated into neural crest stem cellsby culturing them in the presence of triethylenetetraaminetetrahydrochloride 1-100 μM/l for a week. Cells will be thentransdifferentiated into cartilage cells by incubation in a mediumcontaining Transforming Growth Factor Beta (TGF-β; Sigma, St. Louis,Mo.). The new autologous cartilage tissue will be transplanted into apatient.

4. Transdifferentiation of Melanophores into Iridophores

Melanophores will be induced to transform into iridophores by a one-stepprocess using massive amounts (30-100 μg/ml) of guanosine in cellculture or by topical application to the skin or by diet.

5. Transdifferentiation of Melanophores into Neurons

Transdifferentiation of melanosome-containing pigmented cells of thecentral nervous system will enable regeneration of damaged nerves in thecentral nervous system. Melanosome-containing pigmented cells will befirst transdifferentiated into xanthophores using guanosine (1-100μg/ml) and then into neurons using retinoic acid (10⁻⁹-10⁻³ M dependingon source tissue, culture conditions, and type of neurons desired).

Example 11 Treatment of Cancer by Transdifferentiation of MalignantCells to Benign

Cancer (e.g., melanoma or sarcoma) will be treated in a patient byinducing malignant cells to transdifferentiate into benign terminallydifferentiated cells by a two-step process. A battery of standardtransdifferentiating agents will be screened using cultured cancer cellsobtained from a patient, to determine which agent will be the leasttoxic and the most efficient in converting malignant cells into benign(e.g., melanoma cells into benign xanthophores). In a preferred modetransdifferentiating agents such as guanosine (and other purines),copper depletion agents, phenylthiourea, or TPA, in combination withdifferentiating agents such as carotenoids (e.g., beta-carotene,canthaxanthin), retinoids, riboflavin or pteridines, will be tested fortheir ability to transdifferentiate cancer cells and cause growtharrest. The benign phenotype of resulting transdifferentiated cells willbe confirmed using biochemical and immunological methods, as well as byassaying mitotic activity, contact inhibition of growth and metastaticability. Selected transdifferentiating agent(s) will be then applied tothe patient=s tumor topically, by injection, or systemically. Since theindividual patient's melanoma will be used for in vitro screening,treatment of the tumor will be optimized.

Example 12 Induction of Limb Regeneration in Mammals

1. A Method to Increase the Regenerative Potential in the Offspring ofMammals

High regenerative ability in newly born mammals (e.g., rodents, rabbits)will be induced by feeding pregnant females high levels (4-1,000 mg/kg)of transdifferentiating and stabilizing agents, such as guanosine andbeta-carotene, respectively (4-1,000 mg/kg or 1% of diet for eachsubstance). The offspring is expected to possess large numbers oftransdifferentiated or hybrid chromatophores reflecting their increasedregenerative ability. The increased regenerative ability of saidoffspring will be tested using in situ and/or in vitro tissueregeneration assay(s) described in the previous Examples, and comparedto the regenerative ability of the offspring derived from pregnantfemales that were not fed transdifferentiating and stabilizing agents.

2. Regeneration of an Amputated Digit in a Gerbil or Ferret

In gerbils or ferrets that store large amounts of beta-carotene ratherthan primarily converting it to retinol, an amputated digit will beregenerated by feeding the animal a copper deficiency diet with 1%Dunaliella beta-carotene (Henkel Corp., La Grange, Ill.) for 1 monthfollowed by a copper normal diet with beta-carotene as 2% of their dietfor 3 months. A digit (preferentially in an animal without otherdiseases) will be amputated and regrown within months.

3. Regeneration of an Amputated Digit in a Child

A child less then 1.2 years of age (optimally, an infant with no otherdiseases) who has had a digit amputated will regenerate the lost digitby consuming 4 mg/kg of beta-carotene daily for 3 months. The tip of thedigit stump will be surgically opened under local anesthesia first (thetrauma functioning as the destabilizing/differentiating agent) and thedigit will be kept in a chamber at 35-40° C. all day. Bleeding will bestopped. The digit will regenerate in 6 months.

4. Regeneration of an Amputated Appendage in an Adult

An adult (preferably young and without other diseases such as diabetes)with a hand amputated at the wrist will regenerate the missingappendage. Unicellular sebacious glands of Wolff will be isolated from abiopsy taken from the patient=s palm and cultured in vitro. These arecells in the basal layer of the skin of the palms, soles and eyelidswhich contain both premelanosomes and sebaceous droplets. (Wolff,Lancet, 1951, 888-889; Pelfin et al., G. lt. di Derm., 1970, 165:1-5).The cells will be grown to confluency by the addition of a suitablemitogen (such as epidermal growth factor) to the culture medium.Guanosine will be applied topically in an ointment base to the arm stumpwith an occlusive dressing for 3 months. During these 3 months thepatient will be fed 300 mg/day of beta-carotene. Cultured cells will beapplied regularly in high density to the wound surface which will bereopened under local anesthesia. The hand will be regenerated.

Example 13 A Method for Preventing the Progression of AutoimmuneDiseases

The progression of myasthenia gravis (characterized by the presence ofantibodies directed against the acetylcholine receptor in the blood thatcause damage to the patient's muscles) will be prevented by in vitrotransdifferentiation of patient's melanocytes to muscle cells, followedby using these newly generated autologous muscle cells to cleansepatient=s blood of the autoantibodies. The new muscle cells will befirst placed in a membrane or mesh which permits blood to enter butretains the cells. These immobilized cells will be then placed in asterile device similar to a dialysis machine. Once per week the patientwill be dialyzed. The harmful antibodies will adhere to theextracorporeal muscle cells and will not be returned to the body. Inthis way progression of the disease will be prevented.

Example 14 Treatment of Melanoma by Transdifferentiation of Cells into aBenign Phenotype

Materials and Methods

A human melanoma cell line, G-361, was purchased from the American TypeCulture Collection (ATCC catalog number CRL-1424, Manassas, Va.) in acryopreserved state. Upon receipt, the tube containing the frozen cellswas rapidly thawed in a 371 C water bath by swirling the bottom ⅓ of thetube for less than 2 minutes. All subsequent steps were conducted usingstandard methods of sterile technique. The cells were transferred to aT-25 flask containing 5 ml of pre-warmed McCoy=s 5A modified medium(Life Technologies, Rockville, Md.) and incubated at 371 C in ahumidified CO₂ incubator. After approximately 24 hours, the originalmedium was removed and replaced with fresh medium and the cells allowedto grow until they reached approximately 70-80% confluency with themedium being replaced every 2-3 days as needed. At this point, the cellswere subcultured into Leighton tubes at low cell densities using wellknown typsin-EDTA subculturing techniques. After subculturing, the cellswere allowed to re-establish their growth for 48 hours prior toadministration of drugs.

Microscopic observations of the initial human melanoma culture revealeda heterogeneous cell population. Few cells resembled a normal melanocytephenotype. Very few cells displayed detectable amounts of melaninintracellularly. The majority of the cells appeared to be epithelial inmorphology. However, other cell phenotypes were observed in smallernumbers which included fibroblast-type cells, triangular cells and largecells with multiple nuclei. The microscopic phenotypic heterogeneityobserved was compatible with karyotypic analysis provided by ATCCshowing a heterogeneous population with regards to chromosome number.

Later, beta-carotene in tetrahydrofuran was added to McCoy=s 5A modifiedmedium to a final concentration of about 33 mg/ml with a finalconcentration of 0.5% THF. This supplemented medium was added to theLeighton tubes after removal of un-supplemented medium and was replacedevery 2 days during the 7 clay experimental period. At the end of 7days, the cultures were fixed in methanol and the cover slips wereGiemsa stained.

Results

Beta-carotene exerted a dramatic effect on the human melanoma cellcultures. In comparison with control cultures, a large segment of thestarting population died during the experimental period. Of the cellsthat remained, most had triangular morphology and a large nucleus,consistent with neural cells, or were fusiform in shape, consistent withneuroglial or neurilemmal (Schwann) cells. Extensive cell death occurredearly in the 7 day experimental period and the remaining cells did notseem to divide. There were no mitotic figures observed in the remainingcell population.

Conclusions

It is believed that beta-carotene caused transdifferentiation andstabilization of melanoma cells to morphologically benign neurons andneuroglial/neurilemmal cell phentotypes. Those malignant cells whichwere not transdifferentiated into a benign phenotype were killed.

Example 15 Creation of Stem Cells and Transdifferentiation Into Neuronsand Neuroglia or Neurilemmal Cells

Experiment I: Creation of Stem Cells

Methods: The melanoma cell line G-361, described in Example 1.4 abovewas used Guanosine was added to McCoy=s 5A modified medium to a finalconcentration of 3 mg/ml. This supplemented medium was added to theLeighton tubes after removal of unsupplemented medium and was replacedevery 2 days during the 7 day experimental period. At the end of 7 days,the cultures were fixed in methanol and the cover slips were Giemsastained.

Results: During the 7 day experimental period, guanosine inducedphenotypic changes in the overall population such that there were manycells with an increased nuclear to cytoplasmic ratio. There was anincreased number of triangular cells consistent with neural phenotypesand cells whose morphology resembled various types of neuroglial orneurilemmal cells. Many cells exhibiting neuronal morphology wereobserved with two dendritic Ahorns@ on one end of the cells and a longaxonal like projection on the other end. By the end of the 7 dayexperimental period, these cells were frequent in number and readilyobserved. Some examples of all phenotypes present in the initial culturewere also present after the guanosine treatment, at this dose level.

Significance: It is believed that guanosine induced the formation ofstem cells, and that it furthermore stimulated some of these cells tobecome neurons and neuroglia or neurilemmal cells. Factors supportingthe fact that stem cells were created include: the increased number ofcells with a high nucleus to cytoplasm ratio, and, as discussed below,that cells exposed to guanosine and then subsequently to beta-carotenehad a somewhat different response, both morphologically andbehaviorally, than cells only exposed to beta-carotene (and compared tocontrol melanoma cells).

Experiment II: Transdifferentiation into Neurons and Neuroglia orNeurilemmal Cells.

Methods: The melanoma cells line G-361, described in Example 14 abovewas used Guanosine was added to McCoy=s 5A modified medium to finalconcentration of 3 mg/ml. This supplemented medium was added to theLeighton tubes after removal of un-supplemented medium and was replacedevery 2 days for 7 days. Beta-carotene in THF was added to McCoy=s 5Amodified medium at a final concentration of 33 mg/ml with a finalconcentration of 0.5% THF. This supplemented medium was added to theLeighton tubes after removal of guanosine supplemented medium and wasreplaced every 2 days for an additional 7 day experimental period. Atthe end of 14 days, the cultures were fixed in methanol and the coverslips were. Giemsa stained.

Results: During the first 7 days, guanosine produced the effectsdescribed in Experiment 1 above. However, subsequent beta-carotenetreatment did not cause the extensive cell death as occurred withbeta-carotene alone in Example 1A above. Rather, more cells survived,and most of those had a neuronal morphology with a large nucleus,triangular cell body, and processes consistent with dendrites and anaxon. Close observation revealed that the cellular processes present onthe triangular cells, often would be directed towards or touchingadjacent cells forming what appeared to be a loose network between thecells resembling neural networks.

Significance: It is believed that guanosine caused transdifferentiationof the melanocytic cells to stem cells. This is supported not only bytheir morphologic changes, but also that the response of these cells tosubsequent beta-carotene, in terms of greater survival, suggests thattheir phenotype changed. Without wishing to be bound by theory, it isbelieving that beta-carotene further transdifferentiated the stem cellsinto neurons and neuroglial and neurilemmal cells, and then stabilizedthese phenotypes so that these cells established a loose network ofinterconnecting processes resembling neural networks.

Example 16 Transdifferentiation of Normal Melanocytes into Neurons andNeuroglia or Neurilemmal Cells

Dupin, et al. (Proc. Nat. Acad. Sci. USA, 2000, 97: 7882-7887) has shownthat Endothelian 3 was able to induce the conversion of melanocytes intoglial cells. In this Example normal (benign) human melanocytes inculture are obtained from a commercial supplier (Clonetics,Walkersville, Md.). Methods used in Example 14 above are applied toobtain neurons and neuroglia or neurilemmal cells. The phenotypes of theresulting cells are confirmed by electron microscopy andimmunocytochemistry.

Example 17 Transdifferentiation of Normal Melanocytes into AutologousNeurons and Neuroglia or Neurilemmal Cells for Autotransplantation

Normal melanocytes are isolated from a human skin biopsy using standardmethods. The procedures of Example 15 above are applied to produceneurons and neuroglia. Cultures of the resulting cells are put in apharmacologically appropriate vehicle for injection. The resulting cellsare injected into the same individual from which the biopsy is taken.Transplantation is by injection (intraspinal) via lumbar puncture, orintrathecal, or by surgical implantation in nervous tissue. These cellsare able to migrate to areas deficient in neurons or neuroglia orSchwann cells, since neural crest derived cells are highly motile. Theautologous nature of the cells avoids immunological rejection and anyneed for immunosuppression by drugs or other means. This method isapplicable for treatment of mammals (including humans) with diseasessuch as Parkinson=s Disease and Alzheimer=s Disease to replace neuronsthat were destroyed, or in demyelinating diseases such as MultipleSclerosis, where Schwann cells and neuroglia will be replaced.

Example 18 Creating and Regenerating a Developmental Field

I. Steps for Regeneration of a Developmental Field

The universal steps for the regeneration of a developmental field are:

destabilization, transdifferentiation, and stabilization.

When a structure requires regeneration, it is because the structure iseither (1) partially or (2) totally removed.

(1) In the case of (1) above, the arm is amputated at the elbow, thenall elements distal to the elbow (which is in the stump) will have to beregenerated. If the arm is amputated at the wrist, then the entire handwill have to be regenerated.

To regenerate a portion of a developmental field, the stump tissue mustbe stabilized, e.g., by new trauma or by chemicals. Thentransdifferentiation of stump tissues must be accomplished, e.g., bytopical or systemic application of copper depletion agents or diets.Finally stabilization is achieved e.g. by topical or systemicapplication of beta-carotene.

Once the stump tissue is destabilized, the developmental field, by itsnature, intrinsically senses the distalness of the level of amputation.It proliferates and then, concomitant with transdifferentiation,provides the missing structures appropriate for every level to restorethe complete developmental field (e.g., limb).

(2) In the case of (2) above, the lens of the eye will be used as anexample. Complete extraction of the lens leaves no lens material whichmight serve as a stump of like tissue from which the whole lens could bereplaced. The structure and its developmental field are entirelylacking. However, the lens developmental field is a component of the eyedevelopmental field. In this situation the eye developmental field canbe considered the “mother” developmental field because its formationprecedes the formation of the lens developmental field in time and itgives rise to the lens developmental field embryologically. Theremainder of the eye (and its developmental field) remain intact.

In this situation, the regeneration of the lens developmental field isaccomplished by a destabilization, transdifferentiation, andstabilization of tissues from the mother developmental field. Thus thetissues of the iris, which is part of the eye (mother) developmentalfield, undergo the steps of destabilization, transdifferentiation intolens cells, and stabilization of the lens phenotype.

II. Steps for Creation of a Developmental Field

There are a range of situations in which it is desired to create adevelopmental field. For instance, if a child is born without an organor a particular body structure, it would be desirable to create thatstructure. For instance, some children are born with cerebellar aplasia,which is a lack of the cerebellum of the brain which controlscoordination. The developmental field which gives rise to the cerebellumis a component of the brain developmental field. In this case, similarto the case cited above where an entire developmental field must beregenerated, the brain field is the mother developmental field.

The steps for creation of the cerebellar developmental field aresimilarly destabilization and transdifferentiation and stabilization. Inthis application, the posterior (back) of the brain would be exposedsurgically and a small amount of tissue would be traumatized or exposedto a destabilizing chemical. Then a transdifferentiation inducingchemical agent would be topically applied. Finally a stabilizing agentsuch as beta carotene would be included in the diet.

This circumstance is different from the regeneration of a completedevelopmental field cited above, since here the structure never existedin the individual.

Another situation in which it would be desirable to crate adevelopmental field is that of creating supernumerary limbs on a mammal.Such structures can be easily induced in lower animals such asamphibians (Goss, 1969). For instance if a ligature is placed around thelimb of a newt, a fully formed additional limb which is believed to befunctional will sprout at the location of the ligature.

The steps for inducing the formation of it supernumerary structure aredestabilization, trans differentiation, and stabilization of tissues ofthe mother developmental field (e.g. the flank from which the limbextends), or from of the developmental field of the structure itselfsuch as occurs with the newt ligature.

The benefits of functional supernumerary structures such as organs orlimbs are obvious—namely providing additional functional capacity toexisting natural structures.

Example 19 Use of Trauma to Stimulate Destabilization in a Mammal

1. A dog with an inherited retinal degeneration is presented to aveterinarian when the owner notices that it appears to be partiallyblind. The veterinarian makes a small laser burn of the RPE. This isknown to stimulate dedifferentiation and proliferation of RPE cells.Guanosine monophosphate (GMP) is then added to the food at 1 g/kg/day(as a transdifferentiating agent) for a month. When GMP is discontinued,Betatene (Henkel, La Grange, Ill.; 7.5%) is added in the food at 13.5g/day.

2. A dog with a retinal degeneration of unknown etiology is treated withguanosine at 1 g/kg/day for a month (as a destabilizing andtransdifferentiation agent for the RPE) as well as with beta-carotene at1 g/kg/day (as a stabilizing agent). No change is seen in the blindness.The dog is then subjected to a laser burn of the RPE, and the treatmentwith guanosine and beta-carotene is continued. After one month theguanosine is discontinued. Vision is noted to slowly return after thesecond month.

3. Alternatively, a 30 guage needle is introduced just posterior to thelimbus to traumatize (and destabilize) the RPE.

4. Alternatively, sodium iodate, a known RPE toxin (The Retinal PigmentEpithelium, Function and Disease, Wolfensberger and Marmor eds., 1998.Oxford Univ. Press), is injected iv. at 30 mg/kg. This toxic injury tothe RPE causes destabilization of the RPE, and proliferation.

5. To stimulate destabilization of the differentiated state of stumpfibroblasts, nerves, schwann cells, and keratinocytes, an amputee hasthe limb stump surgically opened prior to the use oftransdifferentiation and stabilization agents as above.

6. Alternatively, saturated hypertonic saline soaks of the limb stumpfor twenty minutes, twice per day, for 5 days are performed after theepidermis and collagen scars are debrided initially.

1. A method for treating melanoma in a mammal comprising administeringto a mammal in need of such treatment an amount of aguanosine-containing compound effective to treat melanoma.
 2. The methodof claim 1 further comprising administering an amount of beta-caroteneeffective to treat melanoma.
 3. The method of claim 1 wherein saidguanosine-containing compound is guanosine monophosphate.
 4. The methodof claim 1 wherein said guanosine-containing compound is administeredsystemically.
 5. The method of claim 2 wherein said beta-carotene isadministered systemically.
 6. A method for treating melanoma in a mammalcomprising contacting said melanoma with an amount of guanosinemonophosphate effective to treat melanoma.
 7. The method of claim 6further comprising contacting said melanoma with an amount ofbeta-carotene effective to treat melanoma.
 8. A method for treatingmelanoma in a mammal comprising contacting said melanoma with an amountof adenosine effective to treat melanoma.
 9. The method of claim 8further comprising contacting said melanoma with an amount of retinoicacid effective to treat melanoma.
 10. A method for treating melanoma ina mammal comprising administering to a mammal in need of such treatmentan amount of adenosine effective to treat melanoma.
 11. The method ofclaim 10 further comprising administering an amount of retinoic acideffective to treat melanoma.